Many compounds, either naturally occurring or synthetic, have been found to bind to double stranded nucleic acid, especially double stranded deoxyribonucleic acid (xe2x80x9cdsDNAxe2x80x9d). Depending on their structure, the compounds bind to different parts of the nucleic acid. Some bind to the major groove while others associate with the minor groove. Still others intercalate between adjacent base pairs. Combination binding modes are also known, in which a compound has binding interactions with more than one site in the nucleic acid.
Certain dsDNA binding compounds may be used to regulate the expression of genes for medical purposes. If a disease is characterized by the overexpression or the undesired expression of a gene (e.g., an oncogene), the disease may be treated by suppressing in toto or in part the expression of the gene by the binding of such compounds to the gene or a promoter site thereof. Infections by pathogens such fungi, bacteria, and viruses may be combated with compounds that affect the expression of genes essential for the proliferation of the pathogen.
Whatever the application, the compound must strongly bind to dsDNA, generally meaning that it binds with an association constant of at least 106 Mxe2x88x921, preferably at least about 109 Mxe2x88x921. However, binding strength alone is not determinative of efficacy. Many other factors come into play, including, for instance, cellular uptake, stability, toxicity, binding specificity, and the like. A compound that is acceptable or superior in one characteristic may be fatally deficient in another characteristic. Thus, there is a continuing need to develop new classes of nucleic acid binding compounds for use in such applications.
The present invention provides a new class of compounds, as well as compositions comprising such compounds, methods of synthesizing such compounds, methods of screening such compounds to identify those having anti-infective activity, and methods of using such compounds to prevent or inhibit infections.
In one aspect, the invention provides a class of charged compounds. The members of this class of compounds each comprise a nucleic acid binding moiety, and can be represented by formula (I):
Wxe2x80x94Yxe2x80x94[Het]xe2x80x94Lxe2x80x94[NABM]xe2x80x83xe2x80x83(I)
or a salt thereof, preferably a pharmaceutically acceptable salt. Additionally, esters, amides, prodrugs, isomers, or metabolites of formula I are also within the scope of the present invention.
With respect to this invention, xe2x80x9cNABMxe2x80x9d refers to nucleic acid binding moiety, particularly nucleic acid binding moieties that bind to or associate with double-stranded nucleic acids, particularly dsDNA. NABMs include small molecules, proteins, and nucleic acids. Preferred small molecules include polyamides, particularly synthetic polyamides, and preferred nucleic acids include oligonucleotides. NABMs include intercalating moieties, minor groove binding moieties, major groove binding moieties, and those that include moieties that bind in a combination of such modes, e.g., an NABM that includes both minor and major groove binding moieties.
In formula I above, an NABM is linked to a heteroaromatic moiety (xe2x80x9cHetxe2x80x9d) via linker xe2x80x9cLxe2x80x9d. L represents a bond, preferably a covalent bond, or a linking group. Het represents a heteroaromatic moiety other than N-methyl or N-hydrogen pyrrole, selected from the group consisting of 
wherein one of X1, X2, and X3 is a ring vertex selected from the group consisting of xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, and xe2x80x94NR3xe2x80x94, and the other two of X1, X2, and X3 are ring vertices selected from the group consisting of xe2x95x90Nxe2x80x94 and xe2x95x90CR4xe2x80x94.
Covalently attached to the heteroaromatic moiety [Het] is a substituent having the formula: 
wherein Y is selected from O, S, S(O), SO2, C(R1)2, N(R3)SO2, SO2N(R3) and NR3; and W is halogen or a group having the formula: 
The various R groups in formula I have the following meanings: each R1 is independently selected from H, F, substituted or unsubstituted (C1-C6)alkyl and a substituted or unsubstituted (C1-C6)heteroalkyl group; R2 is a moiety bearing a polar group if Y is other than NR3 and is a moiety bearing a polar group, a substituted or unsubstituted (C1-C12)alkyl group or a substituted or unsubstituted (C1-C12)heteroalkyl group if Y is NR3; each R3 is independently selected from H, a substituted or unsubstituted (C1-C12) alkyl group and a substituted or unsubstituted (C1-C12)heteroalkyl group, provided that neither of R2(R1)2C and R3 contains a 2-chloroethyl or 2-hydroxyethyl group when Y equals NR3; and each R4 is independently selected from hydrogen, halogen, an amino group, a (C1-C8)alkylamino group, a di(C1-C8)alkylamino group, a tri(C1-C8)alkyl ammonium group, a hydroxyl group, a (C1-C8)alkoxy group, a thiol group, a (C1-C8)thioether group, a (C1-C8)sulfone group, a (C1-C8)sulfoxide group, a (C1-C8)sulfonamide group, a substituted or unsubstituted (C1-C12)alkyl group and a substituted or unsubstituted (C1-C12)heteroalkyl group.
Additionally, at least one of R2, [Het], or [NABM] has a positive charge.
In one group of preferred embodiments, the compounds of the invention can be represented by formula (Ia) 
or a salt, preferably a pharmaceutically acceptable salt, or an ester, amide, prodrug, isomer, or metabolite thereof.
In formula Ia, the subscripts a, b, and d are each independently 0, 1, 2, 3, 4, or 5, with the proviso that at least one of a, b, or d is other than 0. The subscripts c, e and f are each independently 0 or 1.
In these embodiments, R5 is selected from halogen, OR7 and N(R7)2. R6 is selected from hydrogen, halogen, a substituted or unsubstituted (C1-C12) alkyl group and a substituted or unsubstituted (C1-C12)heteroalkyl group. Each R7 is independently selected from hydrogen, a substituted or unsubstituted (C1-C12) alkyl group and a substituted or unsubstituted (C1-C12)heteroalkyl group. Each Q is independently selected from xe2x80x94(CH2)2xe2x80x94, xe2x80x94(CH2)3xe2x80x94, and a heteroaromatic ring independently selected from the group consisting of substituted or unsubstituted imidazole, pyrrole, pyrazole, furan, isothiazole, oxazole, isoxazole, thiazole, furazan, 1,2,3-thiadiazole, 1,2,4-thiadiazole, 1,2,5-thiadiazole, 1,3,4-thiadiazole, 1,2,3-triazole, 1,2,4-triazole, 1,2,4-oxadiazole, 1,3,4-oxadiazole, and thiophene rings. Preferably, Q is a thiophene ring. Exemplary suitable substituents in a heteroaromatic ring Q include Cl, F, CH3, and hydroxy.
Compounds according to the invention can be in unpurified, substantially purified, and purified forms. The compounds can be present with any additional component(s) such as a solvent, reactant, or by-product that is present during compound synthesis or purification, and any additional component(s) that is present during the use or manufacture of a compound or that is added during formulation or compounding of a compound.
In another aspect, the present invention provides methods for synthesizing the compounds of the invention. Broadly, such methods comprise linking a NABM to R2(R1)2Cxe2x80x94Yxe2x80x94[Het], either directly or through an optional linking group L. The various moieties of the invention can be synthetic or natural products. Synthetic moieties may be synthesized by solution or solid phase methods. Two or moieties may also be synthesized together.
In yet another aspect, the invention provides compositions comprising a compound according to the invention and one or more excipients, diluents, or carriers. Such compositions can be dry or liquid formulations. The particular composition employed will depend on the intended application for the compound. Compounds according to this invention have been found to be strongly bind dsDNA. Preferably, the association constant for a compound of the invention an dsDNA is at least about 106 Mxe2x88x921, more preferably at least about 109 Mxe2x88x921, and most preferably about 1010 Mxe2x88x921, 1011 Mxe2x88x92, 1012 Mxe2x88x921 or more. Some compositions have been found to be effective in inhibiting the proliferation of pathogens such as fungi and bacteria.
Applications for the compounds and compositions of the invention include anti-infective uses. Such uses can be prophylactic or therapeutic in nature. These uses are accomplished by contacting a pathogen of a eukaryotic organism with an amount of a compound of the invention sufficient to achieve the desired result. Contacting can occur in vitro or in vivo, as the context requires. Preferred embodiments of this aspect involve inhibiting the proliferation of a pathogenic organism. Inhibition can be achieved by killing the organism, by reducing its rate of proliferation, or by reducing or eliminating a pathogenic aspect of the organism, for example, by inhibiting expression of a pathogenic gene (e.g., a gene encoding a toxin). Representative pathogens that can be affected by the preventative and therapeutic methods of the invention include eukaryotic and prokaryotic organisms, as well as viruses. Preferred targets are bacteria and fungi.
The treatment-related aspect of this invention is directed to both animals and plants that serve as hosts, or intermediaries, for the targeted pathogen. As such, the invention has implications in animal health and medicine as well as in agriculture.
In yet another aspect, the invention provides methods of screening to identify compounds of the invention that have anti-infective activity. These screening methods include both in vitro and in vivo screening methods, and can include methods involving an in vitro screen followed by an in vivo screen (e.g., a cell-based screen). In either format, the methods are preferably high throughput methods, meaning that more than about 10, preferably, more than about 100, 1,000, or 10,000 compounds are screened at once. In each of the above recitations, a charged compound refers to compounds that are positively charged under assay or physiological conditions, which are typically neutral or slightly acidic (pH about 5 to about 7). Many compounds are illustrated as having amine components in their neutral form. Nevertheless, one of skill in the art will appreciate that these amines can carry a positive charge (e.g., be protonated) at physiological pH or under typical assay conditions.
These and other aspects and embodiments of the invention are described further in the following detailed description.